One‐Step Preparation of Cell‐Free ATP Regeneration Module Based on Non‐Oxidative Glycolysis Using Thermophilic Enzymes

Abstract

Adenosine triphosphate (ATP) is an essential cofactor for energy‐dependent enzymatic reactions that occur during in vitro biochemical conversion. Recently, an enzyme cascade based on non‐oxidative glycolysis, which uses starch and orthophosphate as energy and phosphate sources, respectively, for the regeneration of ATP from adenosine diphosphate, has been developed (Wei et al., ChemCatChem 2018, 10, 5597–5601). However, the 12 enzymes required for this system hampered its practical usability and further testing potential. Here, we addressed this issue by constructing co‐expression vectors for the simultaneous gene expression of the 12 enzymes in a single expression strain. All enzymes were sourced from (hyper)thermophiles, which enabled a one‐step purification via a heat‐treatment process. We showed that the combination of the two enabled the ATP regeneration system to function in a single recombinant Escherichia coli strain. Additionally, this work provides a strategy to rationally design and control proteins expression levels in the co‐expression vectors.

Synthetic NAD(P)(H) Cycle for ATP Regeneration

“…19,22 For example, a non-oxidative glycolysis pathway using starch as an energy source has been developed for in vivo ATP regeneration. 29 In this work, the simpler NAD(P)(H) cycle was designed to exploit the benefits of a simpler cyclic pathway approach for continuous ATP regeneration.…”

“…Linear pathways are advantageous due to their simplicity; however, it can be challenging to balance substrate concentrations . Cellular metabolisms have evolved many cyclical pathways that serve to both regulate the concentrations of the intermediate metabolites and balance the control of pathway fluxes. , For example, a non-oxidative glycolysis pathway using starch as an energy source has been developed for in vivo ATP regeneration . In this work, the simpler NAD­(P)­(H) cycle was designed to exploit the benefits of a simpler cyclic pathway approach for continuous ATP regeneration.…”

Reaction Engineering and Comparison of Electroenzymatic and Enzymatic ATP Regeneration Systems

“…Various enzymatic systems have been reported to recycle ATP including polyphosphate kinases (PPKs), [5,6] acetate kinase (ACK), [7] pyruvate kinase (PK), [8] creatine kinase (CK), [9] or even by in vitro pathways mimicking glycolysis. [10] The in vitro ATP regeneration by ACK in combination with pyruvate oxidase (POX) has been introduced for cell-free protein synthesis (CFPS) and was recently implemented to a multi-step enzyme cascade. [11,12] The use of POX enables the recycling of inexpensive and stable inorganic phosphate as phosphate donor.…”